New human prostate cell lines were developed from prostatic carcinoma (BRF-41T) and BPH (BRF-55T). Primary cultures were initiated from cellular outgrowths of explanted tissues. A serum-free medium, BRFF-HPC1, was developed for growing human prostatic cancer cells. Cell strains were immortalized with pRSV-T plasmid to generate permanent cell lines that exhibited an epithelial morphology. Both cell lines expressed the epithelial cell markers, cytokeratins 8 and 18 as well as the prostatic marker, PSA, and the androgen receptor gene. They possess the H-ras, K-ras, and p53 genes. We hope that these new human prostatic cell lines will be useful as in vitro models for cancer research.

We developed methodology to isolate and culture rat alveolar Type II cells under conditions that preserved their proliferative capacity, and applied lipofection to introduce an immortalizing gene into the cells. Briefly, the alveolar Type II cells were isolated from male F344 rats using airway perfusion with a pronase solution followed by incubation for 30 min at 37° C. Cells obtained by pronase digestion were predominantly epithelial in morphology and were positive for Papanicolaou and alkaline phosphatase staining. These cells could be maintained on an extracellular matrix of fibronectin and Type IV collagen in a low serum, insulin-supplemented Ham’s F12 growth medium for four to five passages. Rat alveolar epithelial cells obtained by this method were transformed with the SV40-T antigen gene and two immortalized cell lines (RLE-6T and RLE-6TN) were obtained. The RLE-6T line exhibits positive nuclear immunostaining for the SV40-T antigen and the RLE-6TN line does not. PCR analysis of genomic DNA from the RLE-6T and RLE-6TN cells demonstrated the T-antigen gene was present only in the RLE-6T line indicating the RLE-6TN line is likely derived from a spontaneous transformant. After more than 50 population doublings, the RLE-6T cells stained positive for cytokeratin, possessed alkaline phosphatase activity, and contained lipid-containing inclusion bodies (phosphine 3R staining); all characteristics of alveolar Type II cells. The RLE-6TN cells exhibited similar characteristics except they did not express alkaline phosphatase activity. Early passage RLE-6T and 6TN cells showed a near diploid chromosome number. However, at later passages the 6T cells became polyploid, while the 6TN genotype remained stable. The RLE-6T and 6TN cells were not tumorigenic in nude mice. The cell isolation methods reported and the novel cell lines produced represent potentially useful tools to study the role of pulmonary epithelial cells in neoplastic and nonneoplastic lung disease.

Syrian hamster embryo (SHE) cells have a reported culture lifespan limited to 30 to 40 population doublings (PDs). In contrast, we have observed continuous growth of two SHE cell lines without the hallmarks of cellular senescence. Two independent primary isolates (SHE-3, and SHE-15) have been grown for more than 160 PDs (SHE-3) and 50 PDs (SHE-15), respectively, with weekly subcultures and with no sign of senescence. During this study, we observed that SHE-3 cells ceased to multiply after about 30 PDs when grown in a nutritionally-inadequate serum-free medium, but resumed growth when switched to an adequate medium. The chromosomal profiles of both SHE cultures revealed a near diploid karyotype but not a completely normal karyotype, even at early passages with increasing chromosomal changes with continued culture. In addition, Syrian hamster chondrocytes were isolated, partially characterized, and their sensitivity to chemically-induced morphological transformation was compared to that of the standard Syrian hamster embryo (SHE) mixed cell culture. It appears that chondrocytes may be a suitable and more sensitive target for carcinogen-screening in vitro.

The objective of this work was to develop a serum-free medium that supported the growth of secondary Syrian hamster embryo (SHE) cells suitable for use in the morphological transformation assay (MTA). The current assay employs high levels of fetal bovine serum, each lot of which must be selected for its efficacy in the assay. To circumvent the problems associated with the use of serum, two serum-free media (CGM-4 and CGM-9) were developed. The approach used was to replace serum with more defined hormones and growth factors. DME/F12 with low NaHCO3 (pH 6.7) was chosen as a basal nutrient medium since this pH has been reported to give enhanced transformation. Both serum-free media supported the clonal high density growth of normal secondary SHE cells. These media were also found to support the growth of several established SHE cell lines exhibiting successive stages of neoplastic progression. However, significant differential responses of the individual cell lines to CGM-4 and CGM-9 were observed, whereas, in serum-supplemented medium all three lines responded similarly. These results raise the hope that CGM-4 and CGM-9 will be useful for studies of altered autocrine function during transformation and progression as well as for use in the transformation assay itself.

Chromosomal changes in liver epithelial cells undergoing AFB1* induced transformation were studied in two well-characterized epithelial cell cultures derived from rat liver. Diploid liver cells, which did not possess any in vitro transformation marker or the ability to induce tumors in vivo, were treated with the hepatocarcinogen AFB1 and the karyotypes were analyzed sequentially at regular intervals. Although a high percentage of aneuploid cells was seen after AFB1 treatment, no “stem-line” was formed during the period studied. Marker chromosomes appeared prior to the acquisition of malignant potential in liver cells treated repeatedly with AFB1. Chromosomes #1 and #2, were found to be preferentially involved in the formation of marker chromosomes induced by AFB1. Neither marker chromosomes nor malignant potential were noted for 22 weeks after a single treatment with AFB1, to an asynchronous population of rat liver cells. However, when a semi-synchronous population of liver cells were treated once with AFB1, marker chromosomes were observed, but only in a small percentage of the cells. Although the direct implication of the chromosomal alterations in the initiation stage of the carcinogenic process is unclear, procedures known to favor the selective growth of transformed cells in vitro and in vivo resulted in a specific enrichment of the cells with marker chromosomes, suggesting that such cells may be important at least in the progression of the carcinogenic process.

Although methapyrilene (MP) produces hepatocellular carcinomas in rats, it does not elicit many of the cellular responses induced by other hepatocarcinogens. We have investigated the early changes induced in rat liver epithelial cell cultures by MP using morphological, cytochemical, and cytofluorometric techniques. Within 2 h of MP treatment, inclusion bodies which were stainable with lipid stains were observed in the cytoplasm. Ultrastructurally, they resembled lamellar bodies with alternating light and dark lamellae. These bodies were transient in nature, since they disappeared within 24 h of removal of MP. They were, however, retained in the cytoplasm as long as MP was present in the medium. Lamellar bodies appear to be induced in the presence of histamine H1 receptor-blocking agents, since methaphenilene and diphenhydramine produced this reaction, but cimetidine, an H2 antagonist, did not. Morphologically, mitochondria of control cells were long and rod-like, whereas they were short or bizarrely shaped in the MP-treated cells. Moreover, a quantitative increase was observed in the mitochondrial content of the treated cells, when intact liver cells vitally stained with Rhodamine 123 were analyzed with a fluorescence-activated cell sorter. A significant increase in binucleated cells was observed when liver cells were exposed for 10 to 12 days with MP. Collectively, these results suggest that MP might perturb the cytoskeletal elements leading to an alteration in the nuclear and mitochondrial makeup of rat liver cells.

The subcellular and intralobular distributions of a protein which specifically inhibits the proliferation of normal liver cells were determined in rat liver, using a combination of immunological and biochemical techniques. The IgG fraction from an antiserum raised against the hepatic proliferation inhibitor was isolated by protein A-Sepharose CL-4B chromatography and shown to be highly specific for the antigen using electroimmunodiffusion and affinity chromatography. To determine the intracellular location of the inhibitor, subcellular fractions were prepared from adult rat livers by differential centrifugation. The cytoplasmic fraction contained the biologically active cytostatic inhibitor, whereas the nuclear and mitochondrial fractions were inactive. Cytoplasmic localization of the hepatic proliferation inhibitor was further confirmed by anion exchange high performance liquid chromatography and by double immunodiffusion with the anti-hepatic proliferation inhibitor IgG. When liver sections were subjected to histochemical staining mediated through the immune IgG and an avidin-biotinylated horseradish peroxidase complex, the parenchymal liver cells were stained, but endothelial and connective tissue cells were not. Although some staining was evident throughout the liver parenchyma, the most intensely stained cells were located in the centrilobular region. Moreover, an age-dependent increase in the staining intensity and/or in the number of cells containing the proliferation inhibitor was observed. Preliminary experiments showed that little, if any, staining occurred in hepatocellular carcinoma cells. This highly specific IgG can be used to monitor alterations in the content and location of hepatic proliferation inhibitor in proliferative disorders of the liver.

Clonal growth and serial subculture of diploid liver epithelial cells from neonatal rats were achieved in a serum-free medium (SFM) supplemented with linoleic and oleic acid linked to fatty acid-free bovine serum albumin (fafBSA), epidermal growth factor (EGF), transferrin, insulin, selenous acid, and fetuin. Because it is not known whether factors added to defined media facilitate attachment, support proliferation, or both, a serum-free "attachment medium" was first devised in which cells would attach to the substratum without loss of viability. Then a growth medium that would support cell proliferation was developed. Fetuin enhanced the degree of attachment, and the lipid supplements and EGF induced a marked proliferative response. Serum-free medium supported the formation of colonies equivalent in size, number, and morphology to those obtained in serum-supplemented medium. Cells plated at a higher inoculum density and subcultured regularly for up to 25 wk underwent two to three doublings per week and acquired a flattened epithelial cell morphology. Early passages of rat liver epithelial cells, cultured in SFM may be useful in studies of the regulation of cell proliferation and differentiation.

N-Nitroso-N-methylaniline (NMA) and N-nitroso-N-methyl-4-fluoroaniline (p-F-NMA), both non-mutagenic in Salmonella typhimurium and N-nitroso-N-methyl-4-nitroaniline (p-NO2-NMA), a potent mutagen, were tested for carcinogenicity in F344 rats. NMA was shown to induce a high level of tumors in the upper gastrointestinal tract, particularly in the esophagus. Male rats treated with NMA died with tumors at a slightly higher rate than females, although the final tumor yield was the same. Most of the rats treated with p-F-NMA also developed tumors of the esophagus, but they died less rapidly than the NMA treated rats, indicating that p-F-NMA is a slightly weaker carcinogen than NMA. The powerful, directly acting mutagen, p-NO2-NMA did not appear to induce tumors at all since its tumor spectrum was essentially identical to that of the untreated control rats. Thus, the carcinogenic activities of NMA and its substituted analogs do not appear to correlate with bacterial mutagenesis assays. Additionally, NMA, p-F-NMA and N-nitroso-N-methyl-4-bromoaniline, the last a strong mutagen in S. typhimurium, were shown not to induce sister chromatid exchanges in CHO cells and in a clone of a CHO:liver cell hybrid which had previously been shown to be sensitive to chemical agents which require metabolic activation.

The effect of the tumour promoters TPA, phenobarbitone and saccharin on the production of sister-chromatid exchanges (SCE) was studied. TPA produced a small but significant increase of SCE in Chinese hamster cell lines V-79 and CHO, and in a hybrid clone formed by fusion of CHO with rat liver epithelial cells. The enhancement of SCE by TPA was not affected by the type of culture medium, heat-inactivated bovine serum, or the batch of TPA. In the presence of exogenous L-cysteine the enhancement of SCE was reduced. TPA also increase the uptake of 2-deoxyglucose by the cells, to an extent similar to that of the SCE enhancement. This enhancement of SCE by TPA may be explained partly through the formation of free radicals, and partly through alterations in the cell-surface membrane and/or a transient delay in the cell-cycle progression. The other tumour promoters, phenobarbitone and saccharin, also enhanced both SCE and the uptake of 2-deoxyglucose in V-79 cells.

An inhibitor of cell proliferation was purified from rat liver by alcohol precipitation, ultrafiltration, and DEAE-cellulose chromatography. The hepatic proliferation inhibitor was shown to be pure by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, analytical isoelectric focusing, and high-performance liquid chromatography. The hepatic proliferation inhibitor was found to have a molecular weight of 26,000 and an isoelectric point of 4.65. This protein inhibited the proliferation of nonmalignant rat liver cells in culture, and removal of the protein reversed the inhibition produced by low doses. It exerted no effect on the proliferation of malignant rat liver cells.

The induction of sister chromatid exchanges (SCE) by the hepatocarcinogen methapyrilene hydrochloride was investigated using appropriate in vitro and in vivo mammalian cell systems. Methapyrilene, even at the maximum tolerated dose, did not induce SCE in Chinese hamster ovary cells (CHO) or when CHO cells or hamster lung fibroblasts, V-79, were cocultivated with early cultures of rat liver epithelial cells, which are known to metabolize different classes of chemical carcinogens to active forms. Moreover, a hybrid clone of cells (formed by fusion of CHO cells with rat liver epithelial cells), which is highly sensitive to SCE formation by a number of xenobiotics, failed to produce SCE after treatment with methapyrilene. Experiments in vivo with bone marrow cells and in vitro with CHO cells cocultivated with primary hepatocytes from rats also confirmed the inability of methapyrilene to induce SCE in the indicator cells. Since aflatoxin B1 induced SCE in the in vitro and in vivo models, it may be concluded that methapyrilene does not induce SCE at a concentration which is not cytotoxic to the indicator cells in the different systems described. Autoradiographic studies in cultured rat liver cells with tritiated methapyrilene showed that the label was localized in the cytoplasm but not in the interphase nuclei or in the metaphase chromosomes, indicating a lack of interaction of methapyrilene with the nuclear macromolecules of the putative target cells for methapyrilene.

Many short-term carcinogen-screening assays use transformed cells which require exogenous metabolizing systems to activate carcinogens. We have sought untransformed target cell-lines with a high mitotic index and the intrinsic ability to metabolize carcinogens. Hybrid cell clones were established by fusing an untransformed rat liver cell-line (with a normal diploid karyotype, epithelial morphology and carcinogen-metabolizing capacity) and a Chinese hamster ovary cell-line (with a high mitotic index, ability to grow in soft agar, a characteristic of transformed cells, and which cannot metabolize most carcinogens). A number of such clones were characterized with respect to their morphology, karyology, growth properties and the ability to metabolize 7,12-dimethylbenz[a]anthracene. Selected hybrid clones were found to respond to several types of carcinogen, using the induction of sister chromatid exchange as the criterion. A significant increase in the induction was observed in cloned hybrid cell-lines compared with the hamster ovary cell line. Moreover, the same hybrid clones can be used repeatedly without an exogeneous activating system, which makes this test system very practical.